In vitro differentiation and transplantation of ES cells: Mouse ES cell lines expressing green fluorescence protein (GFP) under the control of albumin enhancer/promoter have been established to detect and efficiently isolate (FACS) cells committed to hepatocytic lineage. We have also demonstrated that hepatocyte-like cells can be generated with high efficiency from the ES cells in serum-free defined medium. The ES cell-derived hepatocyte-like cells have been isolated by FACS and characterized using the expression of hepatocyte specific markers. We have now transplanted the ES cell-derived hepatocyte-like cells into isogenic mice (via intra-splenic injection) treated with monocrotaline and CCl4, and shown that the ES-cell derived hepatocytes could be detected in liver tissue after transplantation. These results indicated that mouse ES cell can be directed to differentiate efficiently into hepatocytic lineage, which could in principle provide a potential source of cells for therapeutic transplantations. Transplantation experiments of ES cell-derived hepatocyte-like cells using liver injured mouse models are in progress to determine the efficiency of engraftment and the capacity of ES cell-derived hepatocyte-like cells to repair the damage liver. However, we are in the process of switching our transplantations from the isogenic mice to the Rag deficient mice mainly due to apparent immune incompactability but also with the aim of using the uPA/Rag-/- mouse to test the repopulation capacity of the ES cell-derived hepatocytes. During our transplantation experiments we noted a dramatic difference in the oncogenic capacity between the undifferentiated ES cells (transplanted as a control) and the partially differentiated ES cell derived hepatocyte-like cells. The undifferentiated ES cell quickly homed to the liver after the intra-splenic transplantion, and within a week the liver was packed with teratomas. In constrast the ES cell derived hepatocyte-like cells never developed tumors in the liver. Based on these observations we have hypothesized that during the differentiation of ES cells towards the hepatocytic lineage the oncogenic capacity is lost. Since we can isolate, characterize and transplant the ES cell derivatives at different stages during the differentiation process, we further hypothesized that the transition from the stem cell stage harboring oncogenic potential to an early stage of lineage commitment without (or at least very low) oncogenic potential can be defined. We plan to test this hypothesis and if successful we anticipate that the results will be highly relevant for defining the general phenotypic and genomic characteristics of the "tumor stem cell". Subpopulations of oval cells in rat liver; differentiation and engraftment potential: Adult hepatic stem cells and their early progeny, oval cells, can repopulate and regenerate the diseased liver when the replicative and functional capacities of differentiated hepatocytes are impaired. Although the engraftment and repopulation capacities of unsorted oval cells and small hepatocytes appear to be less efficient than either mature hepatocytes or fetal hepatoblasts. However, little is known about the factors that determine the capacity of hepatic stem cell progenies to engraft in the liver and differentiate into hepatocytes.